The present invention relates to conjugates for mediating cell-specific, compartment-specific or membrane-specific transport of active substances. The invention also relates to methods of producing said conjugates and their use.
As is known, cellular membrane systems are largely impermeable to many substances (e.g. nucleic acids, proteins, chemical substances) which shall be introduced into a cell from outside. For the introduction of nucleic acids it is possible to penetrate cell membranes by physical processes (transfection in the case of eukaryotes, transformation in the case of prokaryotes) and biological processes (infection). In the case of transformation, i.e. the direct take-up of the naked nucleic acid by the cell, the cells are treated beforehand. Various methods are available to produce these xe2x80x9ccompetent cellsxe2x80x9d. Most methods are based on the observations made by Mandel and Higa (J. Mol. Biol. 53, pages 159-163 (1970)) who were the first to show that it is possible to substantially increase the yields occurring when lambda-DNA is taken up by bacteria in the presence of calcium chloride. This method was used successfully for the first time by Cohen et al. (Proc. Natl. Acad. Sci. U.S.A. 69, pages 2210-2114 (1972)) for plasmid DNA and has been improved by many modifications. Another transformation method is based on the observation that high-frequency alternating-current fields can break up cell membranes (electroporation). This technique can be utilized to insert naked DNA not only in prokaryotic cells but also in eukaryotic cell systems (Weaver et al., J. Cell Biochem. 51, pages 426-435 (1993)). Two very mild methods of introducing DNA into eukaryotic cells were developed by Sikes et al. (Hum. Gen. Therap. 5, pages 837-840 (1994)) and Yang et al. (Proc. Natl. Acad. Sci U.S.A. 87, pages 9568-9572 (1990). They are based on the direct injection of the DNA into single cells (microinjection) and on the bombardment of a cell population using microprojectiles of tungsten on the surface of which the corresponding nucleic acid was bound (gene gun), respectively. In a progress parallel to the physical transformation of cells, biological infection methods have proved their efficiency. They comprise in particular the viral introduction of nucleic acids into cells (Chatterjee et al., Science 258, pages 1485-1486 (1992); Cossett and Rusell, Gene Therapy 3, pages 946-956 (1996); Bilbao et al., FASEB J. 11, pages 624-634 (1997)) and the liposome-mediated lipofection (Bennett et al., J. Drug Targeting 5, pages 149-162 (1997)). Reference is also made to standard methods of the liposomal transport (Gao and Huang, Gene Therapy 2, pages 710-722 (1995); Akhtar et al., Nucl. Acid. Res. 19, pages 5551-5559 (1991)) and poly-L-lysine formation (Leonetti et al., Bioconj. Chem. 1(2), page 149 (1990) of active substances to be able to transport them into cells.
Despite the above-listed plurality of methods of passing through the cellular membrane systems, there is no universal method serving for introducing different active substances into cells. All of the above-mentioned physical and biochemical methods are artificial and non-physiological unless they make use of cell-immanent mechanisms. It is presently not yet certain that viruses used as transport vehicles are free of toxicity. They are often not effective and, in addition, they are detected by the immune system.
It was therefore the object of the present invention to provide a possibility of permitting the site-directed and specific introduction of active substances into cells and compartments. The following demands must be complied with in this connection:
universal applicability
cell-specific, compartment-specific and membrane-specific introduction behavior
high degree of effectiveness
low immunogenicity
minimization of the infection risk
sufficiently long residence time.
This object is achieved by the subject matters defined in the claims.
The inventors developed a conjugate comprising the following components:
a transport mediator for the cell membrane (xe2x80x9cPxe2x80x9d),
a cell-specific, compartment-specific or membrane-specific address protein or peptide (xe2x80x9cAPxe2x80x9d), and
an active substance to be transported (xe2x80x9cWxe2x80x9d).
The conjugate according to the invention is preferably composed as follows:
P-AP-W 
More preferably it comprises a spacer (xe2x80x9cSPxe2x80x9d):
P-AP-SP-W 
The transport mediator for the cell membrane (abbreviated as xe2x80x9cPxe2x80x9d above) is a peptide or protein which can penetrate the plasma membrane. The length of this peptide or protein is not subject to limitation as long as it has the above property. Examples of xe2x80x9cPxe2x80x9d are derived preferably from the penetratin family (Derossi et al., 1998, Trends Cell Biol. 8, pages 84-87) or are transportan or parts thereof (Pooga et al., The Faseb Journal (1998), Vol. 12, page 68 et seq.), those of the penetratin family being preferred. An example of xe2x80x9cPxe2x80x9d is a penetratin having the following sequence:
NH2-RQI KIWFQNRRMKWKK-(SEQ ID NO.: 1)
(NH2-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys Trp-Lys-Lys)
Further examples of the transport protein xe2x80x9cPxe2x80x9d are as follows:
Viral transport protein
PTD protein transduction domain (TAT/HIV-1)
1xe2x80x94letter code H2N-YGRKKRRQRRR-COOH (SEQ ID NO: 12)
3-letter code H2N-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-
Bacterial transport molecule
TP protein transport domain TP(Eco)
1-letter code H2N-MTRQTFWHRIKH-CQOH (SEQ ID NO: 13)
3-letter code H2N-Met-Thr-Arg-Gln-Thr-Phe-Trp-His-Arg-Ie-Lys-His
The select xe2x80x9cPxe2x80x9d sequence is produced biologically (purification of natural transport mediator proteins or cloning and expression of the sequence in a eukaryotic or prokaryotic expression system), preferably synthetically, e.g. according to the established Merrifield method (Merrifield, J. Am. Chem. Soc. 85: 2149, 1963).
The selection of the address protein or peptide (abbreviated as xe2x80x9cAPxe2x80x9d above) depends on the membrane or membrane system which has to be penetrated and the target compartment of the cell (cytoplasm, nucleus, mitochondria, chloroplast, endoplasmic reticulum) or the cell organelle which shall be reached. The length of this address peptide or protein is not subject to limitation as long as it comprises the property of ensuring a cell-specific, compartment-specific or membrane-specific transport. For the introduction of active substances, in particular nucleic acids, xe2x80x9cAPsxe2x80x9d are generally used which contain a cell-specific, compartment-specific or membrane-specific recognition signal, directing the attached active substance to its site of action. There are the xe2x80x9cAPsxe2x80x9d to chose from which can transport active substances in the presence or absence of a membrane potential. The pure address sequence is usually sufficient for a transport into the cell compartment. However, it is also possible to chose xe2x80x9cAPsxe2x80x9d which have a cell-specific or compartment-specific peptidase cleavage site. In the most favorable case, this cleavage site lies within the signal sequence but it can also be attached thereto by additional amino acids to ensure the cleavage of the address sequence after the target compartment is reached. The select xe2x80x9cAPxe2x80x9d sequence is produced biologically (purification of natural transport mediator proteins or cloning and expression of the sequence in a eukaryotic or prokaryotic expression system), preferably synthetically, e.g. according to the established Merrifield method (Merrifield, J. Am. Chem. Soc. 85: 2149, 1963). Examples of address proteins or peptides are as follows:
Import into the ER H3N+-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gin-(SEQ ID NO: 2);
Reimport into the ER H2N-Lys-Asp-Glu-Leu-COOxe2x88x92 (SEQ ID NO: 3);
Import into the mitochondria H3N+-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-(SEQ ID NO: 4);
Import into the nucleusxe2x80x94Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO: 5);
H3N+-Pro-Lys-Lys-Lys-Arg-Lys-Val-(=nuclear localisation sequence from 5V40-T antigen) (SEQ ID NO: 6);
Import into peroxisomes H2N-Ser-Lys-Leu-COOxe2x88x92 (SEQ ID NO: 7); and
Binding to the cell membrane H3N+-Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys (SEQ ID NO: 8)
Furthermore, the conjugate may optionally contain a spacer (abbreviated as xe2x80x9cSPxe2x80x9d above) which is preferably located between the address protein/peptide and the active substance to be transported. However, it may also be located additionally or alternatively between the transport mediator and the address protein. The spacer serves for eliminating or positively influencing optionally existing steric interactions between the components. For example, the spacer may be selected from: polylysine, polyethylene glycol (PEG), derivatives of poly-methacrylic acid or polyvinyl pyrrolidone (PVP).
A redox cleavage site, e.g. -cysteine-S-S-cysteine-Oxe2x80x94Nxe2x80x94Hxe2x80x94, is preferably present between the transport mediator and the address protein/peptide. The binding forming between transport mediator and address protein is a redox coupling (mild cell-immanent bond by means of DMSO; Rietsch and Beckwith, 1998, Annu. Rev. Gent 32, pages 163-84):
Cysteine-SH SH-cysteine---- greater than cystine-S-S-cystine
The active substance or active agent (abbreviated as xe2x80x9cWxe2x80x9d above) is not subject to limitations. It can be chosen freely, depending on the effect which shall be produced in a cell. The active substance may be a diagnostic agent and/or a therapeutic agent. The conjugate may also comprise more than one active substance. The active substance may optionally be labeled, e.g. radioactively, with a dye, with biotin/avidin, etc. The active substance may be a nucleic acid, a protein or peptide, a chemical substance, etc. The next ones are mentioned by way of example: cDNA, genomic DNA, complete genes, regulatory elements, transcription factors, molecular probes, oligonucleotides, mRNA, mTRNA, antisense RNA, antisense oligonucleotides, plasmids, viral DNA, synthetic nucleotides, PNA (peptide nucleic acids), single amino acids and their derivatives, peptides, proteins, monoclonal and/or polyclonal antibodies, pharmaceutical active substances, chemotherapeutic agents, dyes, sensitizers, particles.
The conjugate elements xe2x80x9cPxe2x80x9d and xe2x80x9cAPxe2x80x9d are preferably synthesized synthetically according to the Merrifield method (Merrifield, J. Am. Chem. Soc. 85: 2149, 1963). The coupling of the other constituents (e.g. spacer and/or active substance) thereto is made by covalent chemical binding. The redox cleavage site is inserted chemically between xe2x80x9cPxe2x80x9d and xe2x80x9cAPxe2x80x9d by the above-mentioned redox coupling. There is also a covalent bond, preferably an acid amide bond, between an optionally present spacer and the active substance or the address protein and the active substance. Possible alternatives are ether or ester bonds, depending on the functional group(s) present in the substance to be conjugated.
The conjugate is preferably synthesized in the following steps:
1) separate peptide snythesis of xe2x80x9cPxe2x80x9d, xe2x80x9cAPxe2x80x9d and, if applicable, the spacer (e.g. according to the Merrifield method)
2) covalent bond between xe2x80x9cAPxe2x80x9d and active substance, if applicable, with a spacer in between,
3) redox coupling of the product from step 2) with xe2x80x9cPxe2x80x9d by means of redox coupling (e.g. in water/DMSO)
4) purification (e.g. by means of HPLC).
The conjugates according to the invention have the advantage that irrespective of the kind and size of an active substance they can introduce it into cells and transport it into the desired cell compartment. Thus, an improvement of diagnostics and therapy in human and veterinary medicines and an application in scientific research can be anticipated. In partiuclar, the gene therapy can expect a boom on account of the conjugates according to the invention since complete genes including their regulatory elements become transportable. However, all of the other active substances can also be transported more specifically to the site of action by means of the conjugates according to the invention, which reduces the occurrence of undesired side effects. It was found that conjugates up to 25 MDa can be introduced into the cell interior. Moreover, apoptosis is often triggered, which might be a desired effect. The conjugates according to the invention distinguish themselves by a universal usability on account of their cell-specific, compartment-specific and membrane-specific introduction behavior.
The invention is described in more detail by means of the attached figures:
FIG. 1 shows a conjugate according to the invention and includes transport protein RQIKIWFONRRMKWKK-(SEQ ID NO: 1) and nuclear localization sequence PKKKRKV (SEQ ID NO 6)
FIG. 2 shows a general diagram of the Fmoc synthesis;
FIG. 3 shows the results of the fluorescence correlation spectroscopy measurement using AT1 cells
A) conjugate concentration: 50 nM incubation period: 5 hours
B) conjugate concentration: 5 nM incubation period: 5 hours
C) conjugate concentration: 50 nM incubation period: 24 hours
D) conjugate concentration: 5 nM incubation period: 24 hours;
FIG. 4 shows the concentration-dependent and time-dependent transport of rhodamine110(L)-penetratin/RPMI medium; DU145 cells: incubation with 20 xcexcM and 100 pM final concentration;
FIG. 5 shows examples of conjugates according to the invention including:
FIG. 6 shows the production of PNA constructs, wherein the constructs include transport protein RQIKIWFQNRRMKWKK-(SEQ ID NO: 1) and nuclear localization sequence PKKKRKV (SEQ ID NO 6) wherein the active substance was in one case a PNA having the sequence NH2-TAC TGC GAC TCC GG-COOH (anti-sense with respect to rats P2 promoter c-myc=PNAAS) (SEQ ID NO: 10) and then a non-sense (random) sequence having the sequence NH2-TTA AGG AGG CTC-COOH (=PNANS) (SEQ ID NO: 11).
FIG. 7 shows the inhibition of the proliferation of AT-1 cells by introducing an anti-sense construct.
FIG. 8 shows the results of transport into the cytoplasm (Z) or the nucleus (N) for the conjugates produced in Example 1 for incubation periods of 1, 3, 6, 10 and 24 hours.